2.1. RECOMBINANT DNA TECHNOLOGY
Current conventional methods used in recombinant DNA technology involve the insertion of specific DNA sequences into DNA vehicles (vectors) to form recombinant DNA molecules which are capable of replication in a host cell. Generally, though not necessarily, the inserted DNA sequence is foreign to the recipient DNA vehicle, i.e., the inserted DNA sequence and the DNA vector are derived from organisms which do not normally exchange genetic information in nature, or the inserted DNA sequence may be wholly or partially synthetically made. The first step in the recombinant DNA process is the isolation of the DNA sequences which encode the desired gene product. The second step involves the insertion of the isolated genetic sequence(s) into a DNA vector to form a recombinant DNA molecule capable of transforming single-cell hosts. Clones of host cell transformants generate multiple copies of the recombinant DNA vector, thus the inserted DNA sequence is "cloned".
In recent years several general methods have been developed which enable construction of recombinant plasmids. For example, U.S. Pat. No. 4,237,224 to Cohen and Boyer describes production of such recombinant plasmids using restriction enzymes and methods known as ligation. Synthetic oligonucleotides composed of DNA have been employed as linker molecules in cloning DNA. These DNA linker molecules, which can encode restriction endonuclease recognition sequences, are joined to the termini of the DNA molecule to be cloned and thus facilitate insertion into a DNA cloning vehicle (see U.S. Pat. No. 4,321,365).
The recombinant plasmids are then introduced into unicellular organisms by means of transformation. In other words, the recombinant plasmids produced by such conventional methods can be used to transform or "infect" cells in which the vector is compatible, resulting in introduction of the foreign gene into the cell. Because of the general applicability of the techniques described therein, U.S. Pat. No. 4,237,224 is hereby incorporated by reference into the present specification.
Another method for introducing recombinant plasmids into unicellular organisms is described by Collins and Hohn in U.S. Pat. No. 4,304,863 which is also incorporated herein by reference. This method utilizes a packaging/transduction system with bacteriophage vectors.
The recombinant DNA molecules must be capable of autonomous replication in the host cell and should have a marker function which allows for the selection of host cells so transformed. Culturing of the host cell transformants results in host cell replication of the recombinant plasmid and thus generates multiple copies of the DNA sequences. Furthermore, if all of the proper replication, transcription and translation signals are correctly arranged on the plasmid, the foreign gene will be properly expressed in the transformed cells and their progeny.
The controlled bacterial production of such polypeptide products as somatostatin [Itakura, et al., 1977, Science 198: 1056], the component .alpha. and .beta. chains of human insulin [Goeddel, et al., 1979, Proc. Natl. Acad. Sci., U.S.A. 76: 106], and human growth hormone [Goeddel, et al., Nature 281: 544]has already been demonstrated. Production of other polypeptides which are in short supply, as well as viral proteins necessary for vaccine production, are within the capabilities of recombinant DNA technology.
Current methods in gene cloning require that both the gene and the vector exist as double-stranded linear DNA molecules; however, the genetic sequences used for cloning are often isolated in the form of RNA. For instance, when the genes of RNA viruses are sought to be cloned, the entire viral genome is isolated from purified virus, or virus-specific mRNA is isolated from virus-infected cells. Typically, gene sequences are retrieved from eucaryotic cells in the form of messenger RNA (mRNA).
Unlike procaryotic genes, many eucaryotic genes contain intervening sequences (introns) which are not present in the mature mRNA or the corresponding complementary DNA (cDNA) coding for the gene (for review see Chambon, 1981, Scientific American 244(5): 60-71). During mRNA processing, these introns are spliced out of the mRNA prior to translation (for reviews see Crick, 1979, Science 204: 264-271; Sharp, 1981, Cell 23: 643-646).
Isolation of mRNA as the gene source for cloning offers a distinct advantage since expression of eucaryotic genes cloned in procaryotic hosts requires that the coding sequence of the gene of interest be available in a form uninterrupted by intervening sequences. Thus the eucaryotic mRNA sequences, free of introns, are the preferable source of genetic material for cloning in procaryotes.
According to conventional methods, the isolated mRNA or viral RNA is transcribed into complementary DNA (cDNA) copies. The cDNA is then enzymatically processed into double-stranded DNA molecules which can be inserted into cloning vectors.
Such recombinant DNA techniques have been recently used for cloning cDNA copies of RNA viral genomes such as poliovirus (Racaniello and Baltimore, 1981, Proc. Natl. Acad. Sci., U.S.A. 78(8): 4887-4891; Racaniello and Baltimore, 1981, Science 214: 916-919); vesicular stomatitis virus (Rose, 1980, Cell 19: 415-421); and influenza (Sleigh, et al., 1979, Nucleic Acids Res. 7: 879-893); and very recently, double-stranded RNA virus genomes such as rotavirus and reovirus (Imai et al., 1983, Proc. Natl. Acad. Sci., U.S.A. 80: 373-377). These techniques also have an application in the production of viral antigens for use in antiviral subunit vaccines (Heiland and Gething, 1981, Nature (London) 292: 851-852).
One difficulty encountered in cloning the cDNA copy of a gene is in the enzymatic processing of the cDNA molecule into a full length (or nearly full length) double-stranded form. Even if reaction conditions are adjusted to obtain complete cDNA copies of the mRNA, parts of the sequence may be lost during synthesis of the second DNA strand. Second strand synthesis requires the enzyme E. coli DNA polymerase I (Pol I) to utilize the single-stranded cDNA as both primer and template. This results in the formation of a double-stranded DNA molecule which has a single-stranded loop located at the terminus corresponding to the 5' terminus of the original mRNA molecule (Efstratiadis, 1976, Cell 7: 279-288). This single-stranded loop must be digested with S1 nuclease (an enzyme that preferentially degrades single-stranded DNA or RNA) before the double-stranded molecule can be inserted into a cloning vector. The S1 nuclease digestion, however, results in the removal of portions of the cDNA corresponding to the 5'-terminus of the mRNA molecule and reduces the yield of full length cDNA clones. Several modifications of this procedure which eliminate the need for S1 nuclease digestion have been reported (Land, et al.; 1981, Nucleic Acids Res. 9: 2251-2266). More recently, Okayama and Berg (1982, Molecular and Cellular Biology 2(2): 161-170) reported a method for inserting mRNA molecules into DNA cloning vectors which have been modified by oligo(dT) tailing, so that the 3'-poly(A) tail of the mRNA hybridizes to the oligo(dT) tail of the vector. After cDNA synthesis, the free ends of the plasmid and vector are modified to allow hybridization, and finally, ligation.